Understanding the origin of neurotoxicity that leads to neurodegeneration in Alzheimers Disease (AD) is critical to the development of an intervention. Recent studies point to early oligomers of the amyloid beta peptide, A-beta, as critical players in the etiology of AD. Applying traditional approaches to study these oligomers can be challenging because ensemble averaging masks low amounts of transient intermediates and hinders the resolution of species heterogeneity. We propose to apply recently developed approaches, based on single molecule spectroscopy (SMS), that are uniquely suited for these studies to develop the following specific aims: Aim 1: To apply SMS to follow the time evolution of A-beta oligomer formation in solution and to identify those transient oligomers that develop into ordered structures (such as pre- protofibrils) and/or insoluble fibrils. There is clear evidence that during the early stages of A-beta association a highly heterogeneous mix of oligomers develops complicating the identification of the pathological species. We will use SMS of fluorescently labeled A-beta peptides to follow the formation of oligomers as a function of time and to examine the basis for the apparent increased propensity for aggregation of A-beta1-42. The specific hypothesis to be tested is that soluble oligomers form by multiple reaction pathways and that A- beta1-42 more readily forms the initial nucleus for protofibril/fibril formation thus reducing the critical concentration for aggregation in the A-beta 1-42/1-40 mix. Aim 2: To determine the reaction sequence for A- beta oligomer/protofibril formation on the surface of membrane liposomes, to detect membrane permeabilization, to make initial characterization of the pores created and to identify oligomers that affect permeabilization. Recent observations suggest that part of A-beta's neurotoxicity may be associated with membrane binding of pre-fibril structures. We will initiate SMS experiments to test the hypothesis that membranes facilitate the formation of oligomers and to study key aspects of their formation. We will also determine whether a sub-class of the bound A-beta oligomers leads to membrane permeabilization and, if so, at which point this happens and is there a unique species or a multitude of permeabilizing oligomers. Long-term we expect to develop a better understanding of the biomolecular mechanisms and interactions that underlie the evolution of A-beta oligomers, and examine their role in fibril formation and neurotoxicity. [unreadable] [unreadable] [unreadable]